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Method for inducing iPSCs or ESCs to be differentiated into brown fat cells
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A technology of brown fat and cells, which is applied in the fields of biomedicine and biology, can solve the problems of numerous added factors, high cost, and low differentiation efficiency, and achieve the effects of broad clinical application prospects, short culture period, and simple operation
Pending Publication Date: 2019-12-20
NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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Some protocols have shown that compared with adipocytes induced by human ADSVCs, the differentiation efficiency of MSC-like cells derived from human iPSCs or ESCs is relatively low; although infection with lentiviruses carrying key differentiation genes can improve the differentiation of MSC-like cells derived from stem cells. The efficiency of white or brown adipocyte differentiation (still not very high), and the introduction of heterogeneous foreign substances, making its safety not guaranteed
In addition, the culture medium for inducing stem cell-derived MSC-like cells to differentiate into brown adipocytes requires many factors, high cost, and the introduction of heterogeneous foreign substances such as fetal bovine serum, which limits its clinical application value
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Embodiment 1
[0036] Example 1 BAT induction scheme
[0037] 1.1 Culture of human iPSCs and EB formation
[0038] TeSR with Xeno-Free Animal Origin Components TM -E8 TM (STEMCELL Technologies, Catalog#5990) medium cultured human iPSCs in a feeder-free culture mode (take normal human skin fibroblasts, see references Warren L, Ni Y, Wang J, Guo X. Feeder-free derivation of Human induced pluripotent stem cells with messenger RNA. Sci Rep.2012; 2:657. doi: 10.1038 / srep00657. Epub 2012 Sep 14. Acquired by the method of cultivation), the culture condition is 37 degrees, 2.5%-20% O 2 and 10%-15% CO 2 , 95% humidity. The culture method of feeder-free cells is as follows: human iPSCs were cultured in 6-well culture plates coated with human recombinant vitronectin (Vitronectin, STEMCELL Technologies, 07180, the final concentration was 10 μg / ml), and the culture conditions were 37 degrees, Change the medium every day. When subculture, add 300μl Versene Solution (Gibco, CAT#15040066) to the cultur...
Embodiment 2
[0048] 1.1 Culture of human iPSCs and EB formation
[0049] The medium is mTeSR1 (STEMCELL Technologies, Catalog #85850), and the rest are the same as in Example 1.
[0067] 1.5 Induction of Adipogenic Differentiation
[0068] With embodiment 1.
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Abstract
The invention relates to a method for inducing iPSCs or ESCs to be differentiated into brown fat cells. The invention discloses a preparation method of iPSCs-derived MSCs. The method comprises the following steps: cultivating the iPSCs; digesting the iPSCs and performing suspension culture on the digested iPSCs cells to form an embryoid body; adding inducing medium combination A in the suspensionculture process of the embryoid body to induce differentiation; and directly performing adherent culture after the embryoid body is differentiated, or performing adherent culture after digestion or performing sorting culture after digestion to obtain the MSCs. The invention also discloses application of the iPSCs-derived MSCs. The novel method for rapidly preparing the iPSCs-derived MSCs has the characteristics of no foreign matters, suspension culture, shorter culture period, simplicity in operation, infinite amplification and the like, has high efficiency and is more suitable for large-scaleproduction. The iPSCs-derived MSCs accord with the basic characteristic of the MSCs through identification, have a function similar to the marrow-derived MSCs, and can be applied to tissue repair andtreatment on immune-related diseases.
Description
technical field [0001] The invention belongs to the fields of biomedicine and biotechnology, and in particular relates to a method for inducing human iPSCs or ESCs to differentiate into brown fat cells. Background technique [0002] In recent years, the global obese and overweightpopulation has grown rapidly (up to 2.1 billion by 2013), and the obesity situation in my country has become more serious (the population has surpassed the United States to rank first in the world), making obesity an urgent problem in the field of public health. One of the key points. Therefore, it is very necessary to find new, effective and non-toxic means of obesity prevention and treatment in today's day when it is difficult to control obesity with reasonable diet and exercise therapy. As brown adipose tissue (BAT), which has been neglected in the past, has a unique energy consumption mode and strong metabolic capacity, it has entered the field of vision of researchers. In 2009, clinical studi...
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